Transfecting Primary Macrophages with Modified mRNA
Transcription
Begin with a tube containing in vitro transcribed mRNAs that encode green fluorescent proteins.
The mRNA contains modified nucleotides, 5-methyl-cytidine triphosphate, and pseudouridine triphosphate, which prevent immune response and enhance mRNA stability.
The 5'-end of the mRNA is dephosphorylated, preventing its recognition by pattern recognition receptors on macrophages.
Add a cationic lipid-based transfection reagent to the mRNA, facilitating encapsulation of the mRNA through electrostatic interactions and forming a complex.
Introduce the lipid-mRNA complexes into a multi-well plate with adhered mouse-derived primary macrophages and gently agitate for uniform mixing.
The macrophages internalize the mRNA-containing complex into an endosome. Later, the complex fuses with the endosomal membrane, releasing the mRNAs into the cell cytoplasm.
The poly-A tail at the 3'-end stabilizes the mRNA which undergoes protein synthesis to produce green fluorescent proteins.
Under a fluorescence microscope, green fluorescence within the macrophages suggests the successful transfection with modified mRNAs.
